In arthropods, the melanization reaction is associated with multiple host defense mechanisms leading to the sequestration and killing of invading microorganisms. Arthropod melanization is controlled by a cascade of serine proteases that ultimately activates the enzyme prophenoloxidase (PPO), which, in turn, catalyzes the synthesis of melanin. Here we report the biochemical and genetic characterization of a Drosophila serine protease inhibitor protein, Serpin-27A, which regulates the melanization cascade through the specific inhibition of the terminal protease prophenoloxidase-activating enzyme. Our data demonstrate that Serpin-27A is required to restrict the phenoloxidase activity to the site of injury or infection, preventing the insect from excessive melanization.

The prophenoloxidase (proPO) activation pathway, like the vertebrate complement system, consists of a protease cascade and functions as a non-self-recognition system in these animals. Determining the molecular mechanism by which pattern recognition molecules differentiate non-self from self and transduce signals that stimulate defense responses is a key for understanding the ways in which innate immune systems are regulated. However, the proPO system is poorly defined at the molecular level. The proPO-activating system of the insect Holotrichia diomphalia comprises several components, some of which have been cloned and characterized, such as the novel 27-kDa proPO-activating factor-III (PPAF-III) from the plasma of H. diomphalia larvae and two prophenoloxidases. The PPAF-III gene encodes an easter-type serine protease zymogen consisting of 351 amino acid residues with a mass of 40 kDa. The purified 27-kDa PPAF-III specifically cleaved a 55-kDa proPPAF-II to generate a 45-kDa PPAF-II with or without Ca 2؉ present. Furthermore, two Holotrichia prophenoloxidases (proPO-I and -II) have been characterized, and their structural changes during activation were examined by in vitro reconstitution experiments. When the proPOs were incubated with PPAF-I, the 79-kDa proPOs were converted to 76-kDa proPOs, which did not exhibit any phenoloxidase (PO) activity. However, when the proPOs were incubated simultaneously with PPAF-I, proPPAF-II, and PPAF-III in the presence of Ca 2؉ , a 60-kDa protein (PO-1) with PO activity was detected in addition to the 76-kDa proPO-II protein. These results indicate that the conversion of Holotrichia proPOs to enzymatically active phenoloxidase is accomplished by PPAF-I, PAF-II, and PPAF-III through a twostep limited proteolysis in the presence of Ca 2؉ .The prophenoloxidase (proPO) 1 -activating system in invertebrates plays an important role in defense against pathogens and parasites and during cuticular sclerotization. The activation of the proPO system is triggered by elicitors derived from microbial cell walls, such as lipopolysaccharide (LPS), peptidoglycan, and ␤-1,3-glucan (1-3). Several pattern recognition molecules involved in the proPO system, such as peptidoglycan-binding proteins (4), proteins that bind both LPS and ␤-1,3-glucan (5, 6), and ␤-1,3-glucan-binding proteins (7, 8), have been found in various invertebrates. However, the key question is how these pattern recognition molecules can induce activation of the proPO system in response to microbial infection. One hypothesis is that the pattern recognition molecules make a complex with the proPO-activating enzyme(s) and microbial cell wall components, and then activated proPO activating enzyme(s) will convert proPO to active phenoloxidase (PO) by limited proteolysis (1-3).Recently, we characterized two new proPO-activating factors (PPAF-I and PPAF-II) from the coleopteran Holotrichia diomphalia larvae (9, 10). The overall structure of the 37-kDa proPPAF-I is highly similar to that of Drosophila easter, a serine protease that is ...

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